The invention discloses a preparation method of an SERS substrate based on a novel composite material. The preparation method comprises the following steps: S1, synthesizing Au@Ag NPs with a core-shell structure; S2, synthesizing Ag NPs; S3, synthesizing Ag NPs@CoNi-LDH; S4, synthesizing AgNPs@CoNi-LDH@GO: dispersing the Ag NPs@CoNi-LDH prepared in step S3 into ethanol, adding 2 mg/mL of graphene oxide dispersion liquid, heating the mixed solution in a water bath at 60 DEG C, violently stirring for 2 hours, centrifuging, collecting precipitate, washing with ethanol, and re-dispersing in ethanol to obtain ethanol dispersion liquid of the Ag NPs@CoNi-LDH@GO; and S5, preparing the SERS substrate: dispensing the Au@Ag NPs ethanol dispersion liquid prepared in step S1 on a mold, performing air drying, dispensing a layer of Ag NPs@CoNi-LDH@GO ethanol dispersion liquid prepared in step S4, and taking down the mold after natural air drying, thereby obtaining the SERS substrate. The SERS substrate prepared by the preparation method is high in adsorption capacity and can be used for simultaneously and rapidly detecting dimethyl sulfide and 2, 4-di-tert-butylphenol.